Therapeutic antibodies against ror-1 protein and methods for use of same

ABSTRACT

Therapeutic antibodies having binding specificity for ROR-1 expressed on cancer cells (particularly leukemic and lymphomic cells) and pharmaceutical compositions containing one or more such antibodies for use in treating cancer. Methods for diagnosing such cancers through in vitro detection of binding to ROR-1 protein expressed on putative cancer cells are also provided.

BACKGROUND

Tyrosine kinases are important mediators of the signaling cascade,determining key roles in diverse biological processes like growth,differentiation, metabolism and apoptosis in response to external andinternal stimuli. Studies have implicated the role of tyrosine kinasesin the pathophysiology of cancer. Schlessinger J. (2000) Cell,103:211-225; and Robinson et al. (2000) Oncogene, 19:5548-5557.MacKeigan and colleagues used a large-scale RNAi approach to identifykinases that might regulate survival and apoptosis of a human tumor cellline (HeLa), RNAi to ROR1 was found as one of the most potent ininducing apoptosis among the set of RNAi targeting each of 73 differentkinase-encoding genes. MacKeigan et al. (2005) Nat Cell Biol.,7:591-600. However, these investigators did not examine the expressionor function of ROR1 protein in these cells.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII FILE

The Sequence Listing written in file 48537-556C01US_ST25.TXT, createdSep. 27, 2015, 44,093 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated herein by reference in its entirety andfor all purposes.

ROR1, receptor tyrosine kinase like orphan receptor one, is a moleculeexpressed at high levels during embryogenesis that plays a major role inthe development of the skeleton, lungs and nervous system. ROR1expression is greatly decreased in postpartum mammalian cells to levelsthat are barely detectable. ROR1 is a membrane-receptor with anintracellular kinase-like domain and extracellular Frizzled-likecysteine-rich domain, which is common to receptors of members of theWnt-family. ROR1 is member of the ROR family that is evolutionarilyconserved among Caenorhavditis elegans, Drosophila, mice and humans.Wilson C, Goberdhan D C, Steller H. Dror, a potential neurotrophicreceptor gene, encodes a Drosophila homolog of the vertebrate Ror familyof Trk-related receptor tyrosine kinases. Proc Natl Acad Sci USA. 1993;90:7109-7113; Oishi et al. (1997) J Biol Chem., 272:11916-11923;Masiakowski et al. (1992) J Biol Chem., 267:26181-26190; Forrester etal. (2002) Cell Mol Life Sci., 59:83-96; and Oishi et al. (1999) GenesCells, 4:41-56. The actual functional role of the ROR1 protein duringembryogenesis is unknown, although it is believed to be a receptor forWnt proteins that regulate cellular polarity and cell-to-cellinteractions.

Although principally an embryonic protein, ROR1 is expressed uniquely oncertain cancer cells, including in CLL, small lymphocytic lymphoma,marginal cell B-Cell lymphoma, Burkett's Lymphoma, and other cancers(e.g., breast cancers), but not on normal adult tissues and cells. In arecent study, it was found that ROR1, at both mRNA and protein level,was highly expressed in CLL B cells but not normal B cells. Moreover, itwas found that ROR1 is a receptor for Wnt5a, which could induceactivation of NF-κB when co-expressed with ROR1 in HEK293 cells andenhance survival of CLL cells in vitro. This indicates that ROR1 is aCLL survival-signaling receptor for Wnt5a. Another study found that ROR1was expressed in acute lymphocytic leukemia (ALL) as well. Shabani etal. (2007) Tumour Biol., 28:318-326; and Baskar et al. (2008) ClinCancer Res., 14:396-404. Expression of ROR1 protein has now beendemonstrated on a variety of hematologic and solid tumor cancers.

Therapeutic control of ROR1 expression is necessary. However, althoughpolyclonal anti-ROR1 antibodies raised against ROR1 peptide arecommercially available. The inventors developed a monoclonal anti-ROR1antibody, terms 4A5, which reacts with the native ROR1 protein and iscapable of detecting cell-surface expression of ROR1 for flow cytometricanalysis. However, robustly therapeutic antibodies with demonstrableability to inhibit ROR-1 mediated cancer cell proliferation to a degreethat is therapeutically significant for slowing or preventing growth andmetastasis have not been available.

SUMMARY OF THE INVENTION

The invention provides antibodies and combination of antibodies for invivo and in vitro inhibition of ROR-1 cell mediated proliferation ofcells from subjects with cancer, including lymphomas, CLL, smalllymphocytic lymphoma, marginal cell B-Cell lymphoma, Burkett's Lymphoma,renal cell carcinoma, colon cancer, colorectal cancer, breast cancer,epithelial squamous cell cancer, melanoma, myeloma, stomach cancer,brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovariancancer, liver cancer, bladder cancer, prostate cancer, testicularcancer, thyroid cancer, and head and neck cancer, but not in blood orsplenic lymphocytes of nonleukemic patients or normal adults.

The antibodies of the invention are also useful for differentiationbetween ROR1 expressing cancer cells (“ROR1 cancer”) and normal cells.For example, an immunoassay that detects ROR1 in a sample from a subjectby contacting the sample with a ROR1-specific antibody of the inventionand detecting immunoreactivity between the antibody and ROR1 in thesample is provided.

In accordance with a further aspect of the invention, a ROR1 cancer isdiagnosed in a subject by detecting the presence or quantity of ROR1protein in a sample.

The present invention includes compositions that include purified,isolated monoclonal antibodies and combinations thereof that bindspecifically to ROR1 receptor protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of graphs illustrating the results of flow cytometricanalysis of the expansion of CD5+B220low leukemia B cells in ROR1 Tgmice following the adoptive transfer of 1×10⁷ splenocytes from aROR1×TCL1 Tg mouse. Upper panel depicts the expansion from 2 to 4 weeksfollowing adoptive transfer. Percentage of leukemic cells on the contourplot of mCD5 (x-axis) vs mB220 (y-axis) is indicated on above the gateon CD5⁺B220^(low) lymphocytes. Bottom panel depicts the relative ROR1expression (x axis) using the mouse anti-ROR1 4A5 mAb.

FIG. 2 is a diagram outlining the analysis of anti-ROR1 mAb on theadoptive transfer and engragment of ROR1×TCL1 leukemic splenocytes. ROR1Tg mice (4 mice/group) were given 250 ug of 4A5, D10 or control mIgGi.v. on day 0. The following day, 1×10⁷ splenocytes from a ROR1×TCL1 Tgmouse were adoptively transferred i.v. All mice were subsequentlymonitor weekly for expansion of CD5⁺B220^(low) leukemic B cells by flowcytometry beginning at 2 weeks post transfer.

FIG. 3 is a series of graphs illustrating the results of a flowcytometric analysis which demonstrate that anti-ROR1 antibodies of theinvention inhibited the development of CLL-like leukemia in ROR1 Tgmice. 2 weeks after adoptive transfer, the PBMC facs analysis wereperformed. The data showed the anti-ROR1 antibody D10 but not anti-ROR1antibody 4A5 could markedly inhibit the CD5^(dull)B220⁺ andROR1^(bright)B220⁺ leukemic B cell expansion.

FIG. 4 panels A and B show a series of graphs illustrating the resultsof in vivo testing in a murine model of human breast cancer. Theanti-ROR1 antibodies inhibited breast cancer metastasis in rag−/−g−/−deficiency mice. 5E5 MDA-MB-231 breast cancer cell were transferred byi.v. injection to rag−/−g−/− mice on day 1. The rag−/−g−/− deficiencymice were also i.v. injected isotype control or anti-ROR1 antibody (4A5,D10, and 4A5 plus D10) on day 1, 3, 7 and 14 at 100 mg per mice. FIG. 4(center panel) also provides images from IVIS in vivo imaging procedureson the above mice, which were performed every week. 5 weeks later, themice were sacrificed and histology analysis were performed (FIG. 4 panelB). The anti-ROR1 antibody D10 and the antibody combination (4A5 plusD10) both significantly inhibited metastasis of the breast cancer, withinhibition by D10 alone being greater than inhibition by 4a5 alone.

FIG. 5 provides a nucleotide coding sequence comparison of 4A5 Ig heavychain (VH) to the closest germline mouse and human immunoglobulin (Ig)VH.

FIG. 6 provides a nucleotide coding sequence comparison of G6 Ig heavychain (VH) to the closest germline mouse and human immunoglobulin (Ig)VH.

FIG. 7 provides a nucleotide coding sequence comparison of G3 Ig heavychain (VH) to the closest germline mouse and human immunoglobulin (Ig)VH.

FIG. 8 provides a nucleotide coding sequence comparison of H10 Ig heavychain (VH) to the closest germline mouse and human immunoglobulin (Ig)VH.

FIG. 9 provides a nucleotide coding sequence comparison of D10 Ig heavychain (VH) to the closest germline mouse and human immunoglobulin (Ig)VH.

FIG. 10 is a diagram and chart depicting the highly conserved nature ofhuman and murine ROR1.

FIG. 11 is a nucleotide comparison depicting the domain structure andsequence homology of human and murine ROR1 extracellular protein.

FIG. 12 is a chart indicating the extracellular domain which theanti-ROR1 mAbs bind the ROR1 protein.

FIG. 13 is a diagram depicting the chimeric ROR1 proteins generated todetermine the binding domain of each of the anti-ROR1 mAbs.

FIG. 14 is a diagram depicting the truncated ROR1 proteins generated todetermine the sub-regions which each of the anti-ROR1 mAbs binds.

FIG. 15 is a diagram depicting the amino acids which were murinized todetermine residues critical for mAb binding to human ROR1 and a westernblot showing that the 138 glutamic acid residue is critical for antibodyD10 binding to human ROR1.

FIGS. 16A-16B are graphs indicating the K_(D) values for antibody D10(FIG. 16A) and 4A5 (FIG. 16B).

FIG. 17 is a series of graphs illustrating the anti-ROR1 antibody D10 ishighly active in in vivo assays.

FIG. 18 is a diagram outlining the analysis of anti-ROR1 mAb on theadoptive transfer and engragment of ROR1×TCL1 leukemic splenocytes. ROR1Tg mice (5 mice/group) were given 250 ug of 4A5, D10 or control mIgGi.v. on day 0. The following day, 5×10⁵ splenocytes from a ROR1×TCL1 Tgmouse were adoptively transferred i.v. All mice were subsequentlymonitored weekly for expansion of CD5^(dull)B200⁺ leukemic B cells byflow cytometery beginning at 2 weeks post transfer.

FIG. 19 a series of graphs illustrating the results of flow cytometricanalysis of the anti-ROR1 antibodies inhibiting the development ofCLL-like leukemia in ROR1 Tg mice. 2 weeks after adoptive transfer, thePBMC facs analysis were performed. The data showed the anti-ROR1antibody D10 but not anti-ROR1 antibody 4A5 could markedly inhibit theCD5^(dull)B220⁺ and ROR1^(bright)B220⁺ leukemic B cell expansion.

FIG. 20 is a graph illustrating that anti-ROR1 antibody D10 inhibits thedevelopment and expansion of ROR1×TCL1 leukemic B cells in the blood ofrecipient animals until two weeks after receiving the last infusion ofthe mAb.

FIG. 21 is a depiction of the rapid internalization of the anti-ROR1antibody D10 into CLL cells.

FIG. 22 is a series of graphs illustrating the results of flowcytometric analysis showing that anti-ROR1 antibodies D10 and 4A5 areboth internalized into CLL cells. CLL cells were incubated with mouseanti-hROR1 Ab-Alex647 for 30 min at 4° C. Subsequently the cells werewashed and either left at 4° C. or incubated for 4 hours at 37° C.,followed by flow cytometry. The background signal with non-staining isalso shown.

FIG. 23 is a graph illustrating the kinetics of the internalization ofanti-ROR1 antibodies D10 and 4A5.

FIG. 24 is a diagram depicting the amino acids which were murinized todetermine residues critical for mAb binding to human ROR1 and a westernblot showing that the 111 isoleucine residue is critical for antibody4A5 binding to human ROR1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The presently disclosed subject matter are described more fully below.However, the presently disclosed subject matter may be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will satisfy applicable legal requirements. Indeed,many modifications and other embodiments of the presently disclosedsubject matter set forth herein will come to mind to one skilled in theart to which the presently disclosed subject matter pertains having thebenefit of the teachings presented in the foregoing descriptions and theassociated Figures. Therefore, it is to be understood that the presentlydisclosed subject matter is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.

Antibodies of the invention were produced monoclonally using techniquesas previously described. Briefly, Naturally occurring antibodies aregenerally tetramers containing two light chains and two heavy chains.Experimentally, antibodies can be cleaved with the proteolytic enzymepapain, which causes each of the heavy chains to break, producing threeseparate subunits. The two units that consist of a light chain and afragment of the heavy chain approximately equal in mass to the lightchain are called the Fab fragments (i.e., the antigen bindingfragments). The third unit, consisting of two equal segments of theheavy chain, is called the Fc fragment. The Fc fragment is typically notinvolved in antigen-antibody binding, but is important in laterprocesses involved in ridding the body of the antigen.

Because Fab and F(ab′)₂ fragments are smaller than intact antibodymolecules, more antigen-binding domains are available than when wholeantibody molecules are used. Proteolytic cleavage of a typical IgGmolecule with papain is known to produce two separate antigen bindingfragments called Fab fragments which contain an intact light chainlinked to an amino terminal portion of the contiguous heavy chain via bydisulfide linkage. The remaining portion of the papain-digestedimmunoglobin molecule is known as the Fc fragment and consists of thecarboxy terminal portions of the antibody left intact and linkedtogether via disulfide bonds. If an antibody is digested with pepsin, afragment known as an F(ab′)₂ fragment is produced which lacks the Fcregion but contains both antigen-binding domains held together bydisulfide bonds between contiguous light and heavy chains (as Fabfragments) and also disulfide linkages between the remaining portions ofthe contiguous heavy chains (Handbook of Experimental Immunology. Vol 1:Immunochemistry, Weir, D. M., Editor, Blackwell Scientific Publications,Oxford (1986)).

As readily recognized by those of skill in the art, altered antibodies(e.g., chimeric, humanized, CDR-grafted, bifunctional, antibodypolypeptide dimers (i.e., an association of two polypeptide chaincomponents of an antibody, e.g., one arm of an antibody including aheavy chain and a light chain, or an Fab fragment including VL, VH, CLand CH antibody domains, or an Fv fragment comprising a VL domain and aVH domain), single chain antibodies (e.g., an scFv (i.e., single chainFv) fragment including a VL domain linked to a VH domain by a linker,and the like) can also be produced by methods well known in the art.

Monoclonal antibody (mAb) technology can be used to obtain mAbs to ROR1.Briefly, hybridomas are produced using spleen cells from mice immunizedwith ROR1 antigens. The spleen cells of each immunized mouse are fusedwith mouse myeloma Sp 2/0 cells, for example using the polyethyleneglycol fusion method of Galfre, G. and Milstein, C., Methods Enzymol.,73:3-46 (1981). Growth of hybridomas, selection in HAT medium, cloningand screening of clones against antigens are carried out using standardmethodology (Galfre, G. and Milstein, C., Methods Enzymol., 73:3-46(1981)).

HAT-selected clones are injected into mice to produce large quantitiesof mAb in ascites as described by Galfre, G. and Milstein, C., MethodsEnzymol., 73:3-46 (1981), which can be purified using protein A columnchromatography (BioRad, Hercules, Calif.). mAbs are selected on thebasis of their (a) specificity for ROR1, (b) high binding affinity, (c)isotype, and (d) stability.

mAbs can be screened or tested for ROR1 specificity using any of avariety of standard techniques, including Western Blotting (Koren, E. etal., Biochim. Biophys. Acta 876:91-100 (1986)) and enzyme-linkedimmunosorbent assay (ELISA) (Koren, E. et al., Biochim. Biophys. Acta876:91-100 (1986)).

Humanized forms of mouse antibodies can be generated by linking the CDRregions of non-human antibodies to human constant regions by recombinantDNA techniques (see, e.g., Queen et al., Proc. Natl. Acad. Sci. USA86:10029-10033, 1989 and WO 90/07861, each incorporated by reference).Human antibodies can be obtained using phage-display methods (see, e.g.,Dower et al., WO 91/17271; McCafferty et al., WO 92/01047). In thesemethods, libraries of phage are produced in which members displaydifferent antibodies on their outer surfaces. Antibodies are usuallydisplayed as Fv or Fab fragments. Phage displaying antibodies with adesired specificity may be selected by affinity enrichment.

Human antibodies may be selected by competitive binding experiments, orotherwise, to have the same epitope specificity as a particular mouseantibody. Using these techniques, a humanized ROR1 antibody having thehuman IgG1 constant region domain and the human kappa light chainconstant region domain with the mouse heavy and light chain variableregions. The humanized antibody has the binding specificity of a mouseROR1 mAb, specifically the 4A5 mAb described in Examples 4 and 5.

It may be desirable to produce and use functional fragments of a mAb fora particular application. The well-known basic structure of a typicalIgG molecule is a symmetrical tetrameric Y-shaped molecule ofapproximately 150,000 to 200,000 daltons consisting of two identicallight polypeptide chains (containing about 220 amino acids) and twoidentical heavy polypeptide chains (containing about 440 amino acids).Heavy chains are linked to one another through at least one disulfidebond. Each light chain is linked to a contiguous heavy chain by adisulfide linkage. An antigen-binding site or domain is located in eacharm of the Y-shaped antibody molecule and is formed between the aminoterminal regions of each pair of disulfide linked light and heavychains. These amino terminal regions of the light and heavy chainsconsist of approximately their first 110 amino terminal amino acids andare known as the variable regions of the light and heavy chains. Inaddition, within the variable regions of the light and heavy chainsthere are hypervariable regions which contain stretches of amino acidsequences, known as complementarity determining regions (CDRs). CDRs areresponsible for the antibody's specificity for one particular site on anantigen molecule called an epitope. Thus, the typical IgG molecule isdivalent in that it can bind two antigen molecules because eachantigen-binding site is able to bind the specific epitope of eachantigen molecule. The carboxy terminal regions of light and heavy chainsare similar or identical to those of other antibody molecules and arecalled constant regions. The amino acid sequence of the constant regionof the heavy chains of a particular antibody defines what class ofantibody it is, for example, IgG, IgD, IgE, IgA or IgM. Some classes ofantibodies contain two or more identical antibodies associated with eachother in multivalent antigen-binding arrangements.

Fab and F(ab′)₂ fragments of mAbs that bind ROR1 can be used in place ofwhole mAbs. Because Fab and F(ab′)₂ fragments are smaller than intactantibody molecules, more antigen-binding domains are available than whenwhole antibody molecules are used. Proteolytic cleavage of a typical IgGmolecule with papain is known to produce two separate antigen bindingfragments called Fab fragments which contain an intact light chainlinked to an amino terminal portion of the contiguous heavy chain via bydisulfide linkage. The remaining portion of the papain-digestedimmunoglobin molecule is known as the Fc fragment and consists of thecarboxy terminal portions of the antibody left intact and linkedtogether via disulfide bonds. If an antibody is digested with pepsin, afragment known as an F(ab′)₂ fragment is produced which lacks the Fcregion but contains both antigen-binding domains held together bydisulfide bonds between contiguous light and heavy chains (as Fabfragments) and also disulfide linkages between the remaining portions ofthe contiguous heavy chains (Handbook of Experimental Immunology. Vol 1:Immunochemistry, Weir, D. M., Editor, Blackwell Scientific Publications,Oxford (1986)).

With respect to particular antibodies, “specific binding” refers toantibody binding to a predetermined antigen. Typically, the antibodybinds with an affinity corresponding to a K_(D) of about 10⁻⁸ M or less,and binds to the predetermined antigen with an affinity (as expressed byK_(D)) that is at least 10 fold less, and preferably at least 100 foldless than its affinity for binding to a non-specific antigen (e.g., BSA,casein) other than the predetermined antigen or a closely-relatedantigen. Alternatively, the antibody can bind with an affinitycorresponding to a K_(A) of about 10⁶ M⁻¹, or about 10⁷ M⁻¹, or about10⁸M⁻¹, or 10⁹M⁻¹ or higher, and binds to the predetermined antigen withan affinity (as expressed by K_(A)) that is at least 10 fold higher, andpreferably at least 100 fold higher than its affinity for binding to anon-specific antigen (e.g., BSA, casein) other than the predeterminedantigen or a closely-related antigen.

Also, reference to “an antibody having binding specificity for ROR-1protein” includes antibody fragments having at least 90% or 95% sequenceidentity to any of the polypeptide sequences disclosed in SEQ ID NOs: 2.4 6, 8, 12, 14, 16, 18 and 20, including variants modified by mutationto improve the utility thereof (e.g., improved ability to targetspecific cell types and the like). Such variants include those whereinone or more conservative substitutions are introduced into the heavychain and/or the light chain of the antibody.

Such variants include those wherein one or more substitutions areintroduced into the heavy chain nucleotide sequence and/or the lightchain nucleotide sequence of the antibody. In some embodiments thevariant has a light chain and/or heavy chain having a nucleotidesequence at least 80% or at least 90% or at least 95% identical to anyof the nucleotide sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 11, 13,15, 17 and 19.

Polynucleotide sequences which code structural features of theantibodies of the invention include those whose sequences are set forthbelow. Each polynucleotide sequence is followed by the amino acidsequence of the encoded polypeptide. The light chain sequences which areconsidered to be “corresponding” to heavy chain sequences are thoselisted as being for the same antibody; i.e., the F2 heavy chainsequences correspond to the F2 light chain sequences, the D10 heavychain sequences correspond to the D10 light chain sequences, and soforth.

SEQ ID NO: 1 4A5 Mouse Anti-ROR1 mAb Heavy Chain Variable Region CodingSequence:

GAAGTGAAACTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGATTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTAGTCGTGGTGGTACCACCTACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGTCAGGAACATCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGGAAGATATGATTACGACGGGTACTATGCAATGGA CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO: 2 4A5 Mouse Anti-ROR1 mAb Heavy Chain Variable RegionPolypeptide Sequence:

EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQIPEKRLEWVASISRGGTTYYPDSVKGRFTISRDNVRNILYL QMSSLRSEDTAMYYCGRYDYDGYYAMDYWGQGTSVTVSS

SEQ ID NO: 3 4A5 Mouse Anti-ROR1 mAb Light Chain Variable Region CodingSequence:

GACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCCGGACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAAACAGATTGGTTGATGGGGTCCCATCAAGGTTCAGTGGCGGTGGATCTGGGCAAGATTATTCTCTCACCATCAACAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAATTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATGAA AC

SEQ ID NO: 4 4A5 Mouse Anti-ROR1 mAb Light Chain Variable RegionPolypeptide Sequence:

DIKMTQSPSSMYASLGERVTITCKASPDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGGGSGQDYSLTINSLEY EDMGIYYCLQYDEFPYTFGGGTKLEMK

SEQ ID NO: 5 F2, F12 and G6 Mouse Anti-ROR1 mAb Heavy Chain VariableRegion Coding Sequence:

GAGGTCCAGCTACAGCAGTCTGGACCTGAGCTGGAGAAGCCTGGCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTTCGCATTCACTGGCTACAACATGAACTGGGTGAAACAGACCAATGGAAAGAGCCTTGAGTGGATTGGAAGTATTGATCCTTACTATGGTGGTTCTACCTACAACCAGAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAAGAGCCTCACATCTGATGACTCTGCAGTCTATTACTGTGCAAGATCCCCGGGGGGGGACTATGCTATGGA CTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO: 6 F2, F12 and G6 Mouse Anti-ROR1 mAb Heavy Chain VariableRegion Polypeptide Sequence:

EVQLQQSGPELEKPGASVKISCKASGFAFTGYNMNWVKQTNGKSLEWIGSIDPYYGGSTYNQKFKDKATLTVDKSSSTAYMQLKSLTSDDSAVYYCARSPGGDYAMDYWGQGTSVTVSS

SEQ ID NO: 7 F2, F12 and G6 Mouse Anti-ROR1 mAb Light Chain VariableRegion Coding Sequence:

GACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTGTAGGAGAGAGAGTCACTATCACTTGTAAGGCGAGTCAGGGCATTAATAGCTATTCAGGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATTTATCGTGGAAATAGATTGGTGGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAA AC

SEQ ID NOs: 8 F2, F12 and G6 Mouse Anti-ROR1 mAb Light Chain VariableRegion Polypeptide Sequence:

DIKMTQSPSSMYASVGERVTITCKASQGINSYSGWFQQKPGKSPKTLIYRGNRLVDGVPSRFSGSGSGQDYSLTISSLEY EDMGIYYCLQYDEFPYTFGGGTKLEIK

SEQ ID NO: 9 G3 Mouse Anti-ROR1 mAb Heavy Chain Variable Region CodingSequence:

CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGACTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACAACTTCACCAACTACTGGATAAACTGGGTGAAGCTGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAAATTTATCCTGGTAGTGGTAGTACTAATTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGGCATCTGAAGACTCTGCTCTCTATTACTGTGCAAGAGATGGTAACTACTATGCTATGGACTA CTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO: 10 G3 Mouse Anti-ROR1 mAb Heavy Chain Variable RegionPolypeptide Sequence:

QVQLQQPGAELVKPGTSVKLSCKASGYNFTNYWINWVKLRPGQGLEWIGETYPGSGSTNYNEKFKSKATLTADTSSSTAY MQLSSLASEDSALYYCARDGNYYAMDYWGQGTSVTVSS

SEQ ID NO: 11 G3 Mouse Anti-ROR1 mAb Light Chain Variable Region CodingSequence:

GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCACTTGCAGGGCAAGTCAGGACATTAACAATTATTTAAACTGGTATCAACAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAGCATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAACAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAT AAAAC

SEQ ID NO: 12 G3 Mouse Anti-ROR1 mAb Light Chain Variable RegionPolypeptide Sequence:

DIQMTQTTSSLSASLGDRVTITCRASQDINNYLNWYQQKPDGTVKLLIYYTSALHSGVPSRFSGSGSGTDYSLTISNLEQ EDIATYFCQQGNTLPPYTFGGGTKLEIK

SEQ ID NO: 13 D10 Mouse Anti-ROR1 mAb Heavy Chain Variable Region CodingSequence:

CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGACTCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAGTTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGATTCACAAATTATAATTCGGCTCTCAAGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTCTCTTAAAAATGACCAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCCAGGAGAGGTAGTTCCTATTCTATGGACTATTG GGGTCAAGGAACCTCAGTCACCGTCTCCTCA

SEQ ID NO: 14 D10 Mouse Anti-ROR1 mAb Heavy Chain Variable RegionPolypeptide Sequence

QVQLKESGPGLVAPSQTLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGFTNYNSALKSRLSISKDNSKSQVLL KMTSLQTDDTAMYYCARRGSSYSMDYWGQGTSVIVSS

SEQ ID NO: 15 D10 Mouse Anti-ROR1 mAb Light Chain Variable Region CodingSequence:

GAAATTGTGCTCTCTCAGTCTCCAGCCATCACAGCTGCATCTCTGGGCCAAAAGGTCACCATCACCTGCAGTGCCAGTTCAAATGTAAGTTACATCCACTGGTACCAGCAGAGGTCAGGCACCTCCCCCAGACCATGGATTTATGAAATATCCAAACTGGCTTCTGGAGTCCCAGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCATTTATTATTGTCAGCAGTGGAATTATCCTCTTATCACGTTCGGCTCGGGGACAAAGTTGGAAATACAA

SEQ ID NO: 16 D10 Mouse Anti-ROR1 mAb Light Chain Variable RegionPolypeptide Sequence:

EIVLSQSPAITAASLGQKVTITCSASSNVSYIHWYQQRSGTSPRPWIYEISKLASGVPVRFSGSGSGTSYSLTISSMEAE DAAIYYCQQWNYPLITFGSGTKLEIQ

SEQ ID NO: 17 H10 and G11 Mouse Anti-ROR1 mAb Heavy Chain VariableRegion Coding Sequence:

GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCTTCCATTAGTACTGGTGCTAGCGCCTACTTTCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTATTGTGCAAGGATTACTACGTCTACCTGGTACTTCGATGT CTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

SEQ ID NO: 18 H10 and G11 Mouse Anti-ROR1 mAb Heavy Chain VariableRegion Polypeptide Sequence:

EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISTGASAYFPDSVKGRFTISRDNARNILYL QMSSLRSEDTAMYYCARITTSTWYFDVWGAGTTVTVSS

SEQ ID NO: 19 H10 and G11 Mouse Anti-ROR1 mAb Light Chain VariableRegion Coding Sequence:

GACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGTTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAAACAGATTGGTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAGTATGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAA AC

SEQ ID NO: 20 H10 and G11 Mouse Anti-ROR1 mAb Light Chain VariableRegion Polypeptide Sequence:

DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEY EDMGIYYCLQYDEFPYTFGGGTKLEIK

In one aspect, antibodies are provided in which a heavy chain encoded bythe polynucleotide sequence of SEQ ID NO:13 and a light chain encoded bythe polynucleotide sequence of SEQ ID NO:15.

In another aspect, an antibody of the present invention contains a heavychain encoded by the polynucleotide sequence of SEQ ID NO:1 and a lightchain encoded by the polynucleotide sequence of SEQ ID NO:3.

In further aspects, antibodies are provided which have a heavy chainencoded by the polynucleotide sequence of SEQ ID NO: 5 and a light chainencoded by the polynucleotide sequence of SEQ ID NO: 7; or by thepolynucleotide sequence of SEQ ID NO: 9 and a light chain encoded by thepolynucleotide sequence of SEQ ID NO: 11; or by the polynucleotidesequence of SEQ ID NO: 15 and a light chain encoded by thepolynucleotide sequence of SEQ ID NO: 17.

In another aspect, antibodies are provided which contain a heavy chainwith the polypepetide sequence of SEQ ID NO:14 and a light chain withthe polypeptide sequence of SEQ ID NO:16.

In another aspect, antibodies are provided which contain a heavy chainwith the polypeptide sequence of SEQ ID NO:2 and a light chain with thepolypeptide sequence of SEQ ID NO:4.

In one embodiment, isolated polynucleotides which encode an antibodythat specifically binds ROR1 protein are provided which are (a)comprised of a heavy chain region coded by polynucleotides having atleast 90% sequence identity with any of the sequences selected from thegroup consisting of SEQ ID NOs: 1, 5, 9, 13 or 17, (b) comprised of acorresponding light chain region encoded by polynucleotides having atleast 90% sequence identity with any of the sequences selected from thegroup consisting of SEQ ID NOs: 3, 7, 11, 15 or 19, and (c) specificallybinds either the 3′ end or middle portion of the Ig-like region of theextracellular domain of human or murine ROR-1 protein.

Also provided are antibodies which bind residues within the middle ofthe Ig-like region of the extracellular domain of human or murine ROR-1protein (amino acids 1-147 in the human molecule). In one aspect, theantibodies of the present invention bind to amino acids 70-130 of humanROR1. Examples of such antibodies include 4A5, G11, H10 and G3.

Alternatively or additionally, a residue corresponding to the one foundin the extracellular domain of human ROR-1 protein at position 111 iscritical to the binding activity of the antibodies.

Further provided are antibodies that bind residues within the 3′ Ig-likeregion and the linker region between the Ig-like domain and the CRDdomain of human or murine ROR-1 protein (amino acids 1-165 in the humanmolecule). In one aspect, the antibodies of the present invention bindto amino acids 130-165 of human ROR1. Examples of such antibodiesinclude D10, F2, F12 and G6.

Alternatively or additionally, the antibodies bind a glutamic acidresidue corresponding to the one found in the extracellular domain ofhuman ROR-1 protein at position 138.

Alternatively or additionally, a residue corresponding to the one foundin the extracellular domain of human ROR-1 protein at position 138 iscritical to the binding activity of the antibodies.

Alternatively or additionally, the encoded antibody has in vivo activityin reducing leukemic or lymphomic cell burden in an art-accepted animalmodel at a rate of 2-8 times, or at least 2, 3, 4, 5, 6, 7, or 8 times,that of wild-type human anti-ROR1 antibody or monoclonal 4A5 antibody(disclosed herein).

Alternatively or additionally, the encoded antibody has in vivo activityin inhibiting CD5^(dull)B220⁺ and ROR1^(bright)B220⁺ leukemic B cellexpansion.

Alternatively or additionally, the encoded antibody is internalized intoleukemic or lymphomic cells at a rate of at least 2 times, or at least2, 3, 4, 5, 6, 7, 8, 9 or 10 times that of monoclonal antibody 4A5. Suchantibodies are particularly useful as carriers for drug delivery into atargeted cell.

An example of an antibody possessing all of the afore-mentionedfunctional characteristics is D10, which has a heavy chain regionencoded by SEQ ID NO: 13 and a light chain region encoded by SEQ ID NO:15.

In another aspect, polypeptides are provided which consist of orcomprise antibodies which specifically bind ROR1 protein and are (a)comprised of a heavy chain region having at least 90% sequence identitywith any of the sequences of SEQ. ID. NOs: 2, 6, 10, 14 or 18, (b)comprised of a corresponding light chain region having at least 90%sequence identity with any of the sequences of SEQ ID NOs: 4, 8, 12, 16or 20, and (c) specifically binds either the 3′ end or middle portion ofthe Ig-like region of the extracellular domain of human or murine ROR-1protein. In one aspect, the isolated polypeptide is an antibody. In afurther aspect, the polypeptide is a Fab or F(ab)′2.

In certain embodiments, an antibody of the present invention may furthercontain a detectable label. Such labels are known in the art and includeradio-isotopes and fluorescent labels. As such, internalization of acompound evidencing passage through transporters can be detected bydetecting a signal from within a cell from any of a variety ofreporters. The reporter can be a label such as a fluorophore, achromophore, a radioisotope. Confocal imagining can also be used todetect internalization of a label as it provides sufficient spatialresolution to distinguish between fluorescence on a cell surface andfluorescence within a cell; alternatively, confocal imaging can be usedto track the movement of compounds over time. In another approach,internalization of a compound is detected using a reporter that is asubstrate for an enzyme expressed within a cell. Once the complex isinternalized, the substrate is metabolized by the enzyme and generatesan optical signal or radioactive decay that is indicative of uptake.Light emission can be monitored by commercial PMT-based instruments orby CCD-based imaging systems. In addition, assay methods utilizing LCMSdetection of the transported compounds or electrophysiological signalsindicative of transport activity are also employed.

In certain therapeutic embodiments, the selected antibody may beadministered alone, in combination with another antibody of theinvention, or with one or more combinatorial therapeutic agents to treatan ROR-1 cancer. When one or more the antibodies described herein areadministered as therapeutic agents, they may exert a beneficial effectin the subject by a variety of mechanisms. For example, in certainembodiments, antibodies that specifically bind ROR1 are purified andadministered to a patient to neutralize one or more forms of ROR1, toblock one or more activities of ROR1, or to block or inhibit aninteraction of one or more forms of ROR1 with another biomolecule; e.g.,to treat CLL or other ROR1 cancers. All such therapeutic methods arepracticed by delivery of a therapeutically effective dosage of apharmaceutical composition containing the therapeutic antibodies andagents, which can be determined by a pharmacologist or clinician ofordinary skill in human cancer immunotherapy.

In one embodiment, the present invention provides for a method for oftreating cancer by the administration to a human subject in need thereofof a therapeutically effective dose of an antibody according to theinvention.

In another embodiment, the present invention provides a method for oftreating cancer comprising administration to a human subject in needthereof of a therapeutically effective dose of an antibody according tothe invention.

Advantageously, the methods of the invention provide for reduction ofleukemic or lymphomic cell burden (as demonstrated in and equivalent toan art-accepted animal model) of 2-8 times, or at least 2, 3, 4, 5, 6,7, or 8 times, that of wild-type human anti-ROR1 antibody or monoclonal4A5 antibody (disclosed herein).

The methods of the invention further provide a therapeutic approach toinhibiting CD5^(dull)B220⁺ and ROR1^(bright)B220⁺ leukemic B cellexpansion.

As discussed herein, the antibodies of the invention may includehumanized antibodies, and can be combined for therapeutic use withadditional active or inert ingredients, e.g., in conventionalpharmaceutically acceptable carriers or diluents, e.g., immunogenicadjuvants, and optionally with adjunctive or combinatorially activemolecules such as anti-inflammatory and anti-fibrinolytic drugs.Antibodies which readily internalize into cells as demonstrated hereinwith respect to the D10 antibody are also of particular use as carriersfor drug delivery into target cells (for example, as shown in FIGS.21-23). Those of ordinary skill in the art will be familiar with methodsfor producing antibody-drug conjugates useful in such drug deliveryprotocols.

In carrying out various assay, diagnostic, and therapeutic methods ofthe invention, it is desirable to prepare in advance kits comprises acombination of antibodies as described herein with other materials. Forexample, in the case of sandwich enzyme immunoassays, kits of theinvention may contain an antibody that specifically binds ROR1optionally linked to an appropriate carrier, a freeze-dried preparationor a solution of an enzyme-labeled monoclonal antibody which can bind tothe same antigen together with the monoclonal antibody or of apolyclonal antibody labeled with the enzyme in the same manner, astandard solution of purified ROR1, a buffer solution, a washingsolution, pipettes, a reaction container and the like. In addition, thekits optionally include labeling and/or instructional materialsproviding directions (i.e., protocols) for the practice of the methodsdescribed herein in an assay environment. While the instructionalmaterials typically comprise written or printed materials, they are notlimited to such. Any medium capable of storing such instructions andcommunicating them to an end user is contemplated. Such media include,but are not limited to electronic storage media (e.g., magnetic discs,tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.Such media may include addresses to internet sites that provide suchinstructional materials.

In general, an in vitro method of diagnosing a ROR-1 cancer willcomprise contacting putative cancer cells from a human subject with anantibody according to the invention, and detecting binding with ROR-1expressed on said cells as compared to expression on post-embryonichuman non-cancer cells. All such diagnostic methods are practiced bydelivery of a diagnostically effect quantity of antibodies according tothe invention, which can be determined by a diagnostician or in vitrodiagnostic engineer of ordinary skill in human cancer diagnosis.

The following examples are intended to illustrate but not limit theinvention.

Example 1 Generation of Monoclonal Anti-ROR1 Antibodies

For the production of the hybridoma-generated mAbs, mice were inoculatedwith DNA, protein and adenoviral constructs that express theextracellular portion (AA 1-406) of the ROR1 protein that include theIg-like, CRD and Kringle domains and adjacent linker regions (FIGS.10-11). Because of the high degree of homology between the murine andhuman molecules, a variety of cytokines and immune stimulatory agents,such as Freund's Complete Adjuvant, were co-injected to maximize thegeneration of anti-human ROR1 antibodies. Hybridoma-generated mAbs weregenerated and screened for binding to human and murine ROR1. An exampleof hybridoma derived mAbs is D10.

Example 2 Generation of Anti-ROR1 Antibodies Using Phage Display

A second set of antibodies was generated through the use of aproprietary enhanced phage library (Alere, Inc. San Diego). Theseanti-human ROR1 antibodies bind epitopes that span the entire length ofthe extra-cellular domain of the ROR1 protein (FIG. 12). An example of aphage display derived anti-ROR1 antibody is 4A5.

Example 3 In Vitro Analysis of Anti-ROR1 Antibodies

Antibodies generated through either hybridomas or phage display werescreened for binding to human and murine ROR1. It was determined thatthe anti-ROR1 antibodies D10 and 4A5 bound only to human ROR1 and didnot cross react with murine ROR1.

Example 4 Determination of Binding Sites for Anti-ROR1 Antibodies

Because the anti-ROR1 mAbs are species specific, a series of chimericproteins were generated that were used to determine the binding site foreach of the anti-ROR1 mAbs (FIG. 13). As a second level screen, a seriesof deletion constructs were generated to determine the actualextracellular ROR1 domain to which the mAbs bind. Once the bindingdomain was identified, truncated chimeric ROR1 molecules to identifyspecific sub-regions were generated that are recognized by theanti-human ROR1 mAbs (FIG. 14). As a final step, the actual amino acidstargeted by these antibodies were determined. For this final screen,murinized human amino acids in the sub-domain fragments were generatedto determine critical residues required for mAb binding (FIG. 15). Fromthis screening paradigm, the binding sub-domains for the mAbs weredetermined (FIG. 15). It was determine that the D10 anti-human ROR1 mAbrequired the glutamic acid residue at position 138 for binding to theIg-like domain of the human ROR1 molecule. When this amino acid isreplaced with the murine molecule's lysine residue, the D10 molecule nolonger bound to the ROR1 protein.

In a similar manner, it was determined that 4A5 anti-human ROR1 mAbrequired the isoleucine residue at position 111 for binding to humanROR1 molecule (FIG. 24). When this amino acid is replaced with themurine molecule's asparagine residue, the 4A5 molecule no longer boundto the ROR1 protein. It was also determined that the anti-ROR1antibodies G11, H10 and G3 bind the same region as 4A5.

Using standard cross blocking techniques the binding sites for anti-ROR1antibodies F2, F12 and G6 were determined. These experiments determinedthat antibodies F2, F12 and G6 cross block the anti-ROR1 antibody D10,indicating that they share a binding site.

Example 5 Determination of the K_(D) Values for the Anti-ROR1 AntibodiesD10 and 4A5

The K_(D) values for the anti-ROR1 antibodies was determined usingstandard techniques. It was determined that the K_(D) for the D10antibody was 40 nM and for the antibody 4A5 was 4 nM (FIGS. 16A-16B).

Example 6 In Vivo Analysis of Anti-ROR1 Antibodies

The D10 mAb was assessed in several in vivo models. In a murine in vivoxenograph, niche-dependent, activity model two doses of the mAb wereadministered at 10 mg/kg against 4 primary patient CLL cells in 76 mice.As shown in FIG. 17, D10 mAb substantially eliminated patient CLL cellsin a dose dependent manner. In contrast, the 4A5 mAb had minimalactivity in these studies even though the kDa of this mAb is 10 foldgreater (4 vs. 40) for the D10 mAb.

In addition to this activity model, the D10 mAb was also tested in animmune competent transgenic mouse model that spontaneously generatesleukemic cells expressing the human ROR1 protein (FIGS. 18-20). TheROR1-specific mAbs D10 and 4A5 or control IgG antibodies (10 mg/kg) wereadministered before and after adoptive transfer of ROR1×TCL1 CLL B cellsinto Balb C mice. The D10 mAb, but not control IgG or 4A5, was able toinhibit the development and expansion of the ROR1×TCL1 leukemic B cellsin the blood of recipient animals until two weeks after receiving thelast infusion of MAb.

Along with the anti-leukemic activity of this mAb, it has also beenshown that the D10 anti-ROR1 antibody is internalized into patient CLLcells and B cell leukemia and lymphoma cell lines at a greater rate anddegree than other anti-ROR1 MAbs that bind other antigenic sites on theextracellular portion of the ROR1 protein (FIGS. 21-23). Because of theabsence of the ROR1 protein on post-partum tissues and its rapid rate ofinternalization, the D10 mAb may serve as an excellent carrier proteinfor drugs; for example, for use in directed antibody-drug conjugate(ADC) mediated cytotoxicity. Based on these preclinical findings, theD10 mAb has potential to have therapeutic activity against ROR1expressing leukemias, lymphomas and solid tumor cancers as a targetedtherapy and/or conjugated drug carrier.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

1.-27. (canceled)
 28. A method of identifying an anti-ROR1 antibody, themethod comprising: (i) contacting an antibody with a first ROR1polypeptide comprising an isoleucine at a position corresponding toposition 111 of SEQ ID NO:36; (ii) detecting said antibody binding tosaid first ROR1 polypeptide; (iii) contacting said antibody with asecond ROR 1 polypeptide not comprising an isoleucine at a positioncorresponding to position 111 of SEQ ID NO:36; and (iv) detecting saidantibody not binding to said second ROR1 polypeptide, therebyidentifying an anti-ROR1 antibody.
 29. The method of claim 28, whereinsaid second ROR1 polypeptide comprises an asparagine at a positioncorresponding to position 111 of SEQ ID NO:36.
 30. The method of claim28, wherein said first ROR1 polypeptide is a truncated ROR1 polypeptide.31. The method of claim 30, wherein said truncated ROR1 polypeptidecomprises amino acid residues 1-165 of SEQ ID NO:36.
 32. The method ofclaim 28, wherein said second ROR1 polypeptide is a truncated ROR1polypeptide.
 33. The method of claim 32, wherein said truncated ROR1polypeptide comprises amino acid residues 1-165 of SEQ ID NO:37.
 34. Themethod of claim 28, wherein said antibody is a humanized antibody. 35.The method of claim 28, wherein said antibody is an antibody fragment.36. The method of claim 28, wherein said antibody is a chimericantibody.
 37. The method of claim 28, wherein said antibody is a singlechain antibody.
 38. The method of claim 28, wherein said antibody bindsto said first ROR1 polypeptide with a K_(D) of less than about 4 nM.